Research ArticleANEMIA

Vasopressin stimulates the proliferation and differentiation of red blood cell precursors and improves recovery from anemia

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Science Translational Medicine  29 Nov 2017:
Vol. 9, Issue 418, eaao1632
DOI: 10.1126/scitranslmed.aao1632
  • Fig. 1. Anemia in patients with central diabetes insipidus.

    (A) Ninety-two patients who had central diabetes insipidus were identified at the National Institutes of Health Clinical Center: 45 women (red circles) and 47 men (blue squares). Their hemoglobin, hematocrit, and red blood cell distribution width (RDW) values were averaged for all the measurements made and are depicted by a single, thin red or blue line on the graph. The black line on each graph represents the average values in healthy subjects. The hematocrit and hemoglobin values of these patients are significantly (***P < 0.0001) different from the general U.S. population. (B) Reverse transcription polymerase chain reaction (RT-PCR)–based detection of AVPR1A, AVPR1B, and AVPR2 mRNAs in human CD34+ cells. Lane 1, 100–base pair (bp) ladder; lane 2, RT-PCR product; lane 3, no template. (C) Human CD34+ cells on day 3 of two-phase liquid cultures (immunocytochemistry). All three arginine vasopressin (AVP) receptors are present. AVPR1B appears to be much more abundant than AVPR1A and AVPR2. Scale bars, 10 μm. (D) AVPR1A, AVPR1B, and AVPR2 receptor proteins during erythroid differentiation in two-phase liquid culture (Western blotting). (E) AVP and d(Cha4)-AVP (an AVPR1B agonist) increase intracellular Ca2+ in human hematopoietic stem and progenitor cells (HSPCs).

  • Fig. 2. Erythroid differentiation of human CD34+ cells promoted by AVP.

    (A) Both AVP and AVPR1B agonist cause HSPCs (CD34+) to proliferate in vitro. This effect was blocked by a specific AVPR1B antagonist. Data and error bars are means + SEM; ns, not significant. *P < 0.05. OD 450, optical density at 450 nm. (B) Colony-forming assays were conducted to determine burst-forming unit–erythroid (BFU-E) and colony-forming unit–erythroid (CFU-E) colony numbers when CD34+ cells were exposed to AVP or a specific AVPR1B agonist added to the methylcellulose. AVP and the AVPR1B agonist significantly increased BFU-E colonies but not CFU-E. n = 3 independent donors and two replicates each. Data and error bars are means + SEM. *P < 0.05. (C) Colony-forming assays were conducted after 48 hours of treatment with AVP or AVPR1B agonist. Both AVP and AVPR1B agonist significantly increased CFU-E, but only the AVPR1B agonist increased BFU-E colonies. n = 2 independent donors and two replicates each. Data and error bars are means + SEM. *P < 0.05, ***P < 0.001. (D) May-Grünwald/Giemsa staining of cells from BFU-E colonies. Cells were harvested by Cytospin after growing for 10 days in control medium or medium with 10 nM AVP. Most of the cells in the control colonies are basophilic erythroblasts. Addition of AVP seems to accelerate differentiation; many reticulocytes can be seen, indicated by the arrowhead. The asterisk is placed between two erythrocytes that are in the process of enucleation to become reticulocytes. Scale bars, 100 μm (low magnification; left) and 10 μm (high magnification; right). (E) Human CD34+ cells were cultured with or without AVP. The cells were counted every 3 days. Each graph represents one donor. (F) Western blots of p-STAT5 and signal transducer and activator of transcription 5 (STAT5). Human CD34+ cells were challenged with erythropoietin (EPO), AVP, or EPO + AVP on days 9 and 12. On day 9, the effect of EPO + AVP on STAT5 phosphorylation was markedly greater than the effects of EPO or AVP alone. On day 12, the late-stage erythroid cells were more responsive to AVP than EPO, and the two agents no longer appeared to act in concert. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 3. Recovery of AVP-deficient rats from anemia induced by sublethal irradiation.

    AVP-deficient Brattleboro and control Long-Evans rats were subjected to sublethal irradiation. Hematocrit (A), corrected reticulocyte percentage (calculated by multiplying the reticulocyte percentage by the ratio of measured hematocrit and normal hematocrit) (B), and high-intensity reticulocyte percentage (C) were measured on the days indicated. The recovery of Brattleboro rats was significantly slower than the recovery of control Long-Evans rats. Data and error bars are means + SD. Brattleboro (n = 6), ***P < 0.001; Long-Evans (n = 9), ***P < 0.001.

  • Fig. 4. The effects of AVP or d(Leu4Lys8)VP, a specific AVPR1B agonist, on anemic rodents.

    (A) Avpr1a, Avpr1b, and Avpr2 receptor expression in fluorescence-activated cell–sorted mouse bone marrow (BM) linSca-1+c-kit+ (LSK) cells. Lane 1, 100-bp marker; lane 2, RT-PCR product; lane 3, no template. (B) Flow cytometry sorting of mouse bone marrow erythroid lineage cells using CD44 and TER119 markers into four populations. Population I, proerythroblast; population II, basophilic; population III, polychromatic; population IV, orthochromatic. Numbers represent the percentage of total TER119-positive cells. n = 4 mice, C57BL/6. (C) Gene expression changes of Avpr1a, Avpr1b, and Avpr2 receptors in four different populations of bone marrow erythroid lineage cells. Error bars represent SEM; n = 4 mice, C57BL/6. TBP, TATA box–binding protein. (D) C57BL/6 mice were injected with AVP (100 μg/kg), and after 16 hours, the erythroid progenitors in the bone marrow and spleen were analyzed with flow cytometry. Data and error bars are means + SD; n = 5 mice per group; **P < 0.01. (E) C57BL/6 mice were hemorrhaged and injected once with AVP (100 μg/kg) or AVPR1B agonist (100 μg/kg). Hematocrit and corrected reticulocytes were measured on the days indicated. After hemorrhage, both AVP and d(Leu4Lys8)VP (AVPR1B agonist) significantly improved recovery times. Data and error bars are means + SD; n = 6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. (F) C57BL/6 mice received whole-body irradiation (440 centigrays; two times within 12 hours) and were injected with AVP (100 μg/kg). Hematocrit and corrected reticulocytes were measured on the days indicated. After irradiation of the bone marrow, AVP administration resulted in a significantly higher hematocrit and reticulocyte values. Data and error bars are means + SD; n = 6 mice per group. *P < 0.05, ***P < 0.001. (G) C57BL/6 mice were splenectomized and allowed to recover for 1 month. The mice were subjected to hemorrhage and immediately injected with AVP (100 μg/kg) or AVPR1B agonist (100 μg/kg). Hematocrit and corrected reticulocytes were measured on the days indicated. In splenectomized mice, AVP still promoted recovery from anemia. Data and error bars are means + SD; n = 6 mice per group. **P < 0.01; ***P < 0.001. (H) Mice were sublethally irradiated and then given either wild-type (WT) or Avpr1b-deficient bone marrow. Two months later, they had 10 to 20% of their blood withdrawn or were treated with phenylhydrazine. Four days after hemorrhage, there was a significant increase in blood-corrected reticulocyte counts in animals given wild-type versus receptor-deficient bone marrow. This was not reflected in an increase in hematocrit. Six to 14 days after the phenylhydrazine treatment, corrected reticulocyte counts were significantly elevated in mice given wild-type bone marrow versus receptor-deficient bone marrow. On day 6, the hematocrit of the wild-type mice was also higher. Data and error bars are means + SD; n = 6 mice per group. *P < 0.05, **P < 0.01. (I) C57BL/6 mice were hemorrhaged (this sample was considered as the baseline) and injected with phosphate-buffered saline (PBS) or EPO (50 U/kg) or AVP (100 μg/kg). Hematocrits were measured at the time points indicated. AVP treatment significantly increased hematocrit by 12 hours compared to EPO or vehicle (PBS) treatment. Error bars represent SD; n = 6 mice per group. **P < 0.01, ***P < 0.001. (J) C57BL/6 mice were hemorrhaged and injected with either 100 μg of EPO-neutralizing antibody (Ab) or its isotype (ISO) control, with or without AVP (100 μg/kg). Hematocrits were measured at the time points indicated. Data and error bars are means + SD; n = 5 mice per group. *P < 0.05, ***P < 0.001.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/418/eaao1632/DC1

    Fig. S1. AVP agonists affect intracellular calcium, and AVP increases cAMP in human progenitors.

    Fig. S2. Norepinephrine and AVP induce an increase in the number of BFU-E colonies.

    Fig. S3. AVP and AVPR1B agonist [d(Cha4)-AVP] increase BFU-E formation.

    Fig. S4. Band intensities from Western blots were analyzed by ImageJ.

    Fig. S5. Response to anemia is coordinated by AVP and EPO.

    Table S1. RBC indices for patients (provided as an Excel file).

    Table S2. Human RT-PCR primers.

    Table S3. Mouse RT-PCR primers.

    Table S4. Mouse real-time PCR primers.

    Table S5. Antibodies used for flow cytometry.

  • Supplementary Material for:

    Vasopressin stimulates the proliferation and differentiation of red blood cell precursors and improves recovery from anemia

    Balázs Mayer, Krisztián Németh, Miklós Krepuska, Vamsee D. Myneni, Dragan Maric, John F. Tisdale, Matthew M. Hsieh, Naoya Uchida, Heon-Jin Lee, Michael J. Nemeth, Kenn Holmbeck, Constance Tom Noguchi, Heather Rogers, Soumyadeep Dey, Arne Hansen, Jeffrey Hong, Ian Chow, Sharon Key, Ildikó Szalayova, Jerome Pagani, Károly Markó, Ian McClain-Caldwell, Lynn Vitale-Cross, W. Scott Young, Michael J. Brownstein, Éva Mezey*

    *Corresponding author. Email: mezeye{at}mail.nih.gov

    Published 29 November 2017, Sci. Transl. Med. 9, eaao1632 (2017)
    DOI: 10.1126/scitranslmed.aao1632

    This PDF file includes:

    • Fig. S1. AVP agonists affect intracellular calcium, and AVP increases cAMP in human progenitors.
    • Fig. S2. Norepinephrine and AVP induce an increase in the number of BFU-E colonies.
    • Fig. S3. AVP and AVPR1B agonist [d(Cha4)-AVP] increase BFU-E formation.
    • Fig. S4. Band intensities from Western blots were analyzed by ImageJ.
    • Fig. S5. Response to anemia is coordinated by AVP and EPO.
    • Table S2. Human RT-PCR primers.
    • Table S3. Mouse RT-PCR primers.
    • Table S4. Mouse real-time PCR primers.
    • Table S5. Antibodies used for flow cytometry.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. RBC indices for patients (provided as an Excel file).

    [Download Table S1]

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