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Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin.

ABSTRACT: Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2-/- renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2-/- mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation.

SUBMITTER: Kobayashi H

PROVIDER: S-EPMC4855934 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin.

Kobayashi Hanako H Liu Qingdu Q Binns Thomas C TC Urrutia Andres A AA Davidoff Olena O Kapitsinou Pinelopi P PP Pfaff Andrew S AS Olauson Hannes H Wernerson Annika A Fogo Agnes B AB Fong Guo-Hua GH Gross Kenneth W KW Haase Volker H VH

The Journal of clinical investigation 20160418 5

Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell ...[more]

PMID: 27088801

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Project description:Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1-/-, or Sphk2-/- mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2-/- renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2-/- cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.

Project description:IntroductionMesenchymal stem cells (MSCs) play a central role in the remediation of cell and tissue damage. Erythropoietin (EPO) may enhance the beneficial influence of MSCs during recovery from tissue and organ injuries. Microvesicles (MVs) released from MSCs contribute to the restoration of kidney damage. We studied the influence of EPO on MVs derived from MSCs, and the protective effects of these factors in subjects with chronic kidney disease (CKD).MethodsThe MVs derived from untreated MSCs (MSC-MVs) or from MSCs incubated in different concentrations of EPO (1, 10, 100, and 500 IU/ml EPO-MVs) were used to treat renal injury of unilateral ureteral obstruction (UUO) in vivo, and transforming growth factor-β1 (TGF-β1)-induced fibrosis in a human renal proximal tubular epithelial (HK2) cell line in vitro. Western blot and reverse transcription polymerase chain reaction (RT-PCR) analyses were used to evaluate the expression of epithelial and mesenchymal markers in the renal tissue and HK2 cells. Flow cytometry was used to assess apoptosis within the HK2 cells, and microRNA (miRNA) microarray assays were used to determine the expression profiles of miRNA in the MSC-MVs and EPO-MVs.ResultsCompared to MSC-MVs (untreated), there was a significant increase in the number of EPO-MVs derived from MSCs treated with 1-100 IU/ml EPO, and these EPO-MVs had a greater benefit in UUO mice on days 7 and 14. Moreover, the EPO-MVs had a better restorative effect following TGF-β1-induced fibrosis in HK2 cells at 24 h and 48 h. The flow cytometry results revealed that both types of MVs, especially EPO-MVs, play an important anti-apoptotic role in HK2 cells treated with TGF-β1. The miRNA profiles of the MVs revealed that EPO-MVs changed 212 miRNAs (fold-change ≥ 1.5), including miR-299, miR-499, miR-302, and miRNA-200, and that 70.28 % of these changes involved upregulation. The changed miRNA in EPO-MVs may have contributed to their enhanced protective effects following renal injury compared to MSC-MVs.ConclusionsThere was a dose-dependent increase in the level of EPO-MVs within the range of 1-100 IU/ml EPO. Although both MSC-MVs and EPO-MVs protect the kidney from fibrosis-related damage, there is a superior effect of EPO-MVs.

Project description:BackgroundForkhead transcription factors control cell growth in multiple cancer types. Foxd1 is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported.MethodsTranscriptome data from the TCGA database was used to correlate FOXD1 expression with patient survival. FOXD1 was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of the FOXD1 pathway with cell cycle progression was investigated through correlation analysis using the TCGA database.ResultsFOXD1 expression level in ccRCC correlated inversely with patient survival. Knockout of FOXD1 in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-OFOXD1null. In vivo, xenografted 786-OFOXD1null showed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-OFOXD1null had an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-OFOXD1null, and increased DNA damage. Genes correlate with FOXD1 in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation.ConclusionsWe show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression of FOXD1 and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.

Dataset Information

Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin.

Publications

Distinct subpopulations of FOXD1 stroma-derived cells regulate renal erythropoietin.

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