HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/14/2023 05-3820fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kato, H. Articles by Fukamizu, A. Search for Related Content PubMed PubMed Citation Articles by Kato, H. Articles by Fukamizu, A.

Enhanced erythropoiesis mediated by activation of the renin-angiotensin system via angiotensin II type 1a receptor

Hideki Kato^*,||, Junji Ishida^*, Shigehiko Imagawa, Tomoko Saito^*, Norio Suzuki^*,, Toshiki Matsuoka^*, Takeshi Sugaya^*, Keiji Tanimoto^*, Takashi Yokoo^¶, Osamu Ohneda^*,, Fumihiro Sugiyama, Ken-ichi Yagami, Toshiro Fujita^||, Masayuki Yamamoto^*,, Masaomi Nangaku^|| and Akiyoshi Fukamizu^*,1

^* Center for Tsukuba Advanced Research Alliance (TARA),
Institute of Basic Medical Sciences,
Division of Hematology, Institute of Clinical Medicine,
Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki, Japan;
^|| Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, Hongo, Bunkyo, Tokyo, Japan; and
^¶ Division of Nephrology and Hypertension, Department of Internal Medicine, Jikei University School of Medicine, Nishi-Shimbashi, Minato-ku, Tokyo, Japan

¹Correspondence: Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. E-mail: akif{at}tara.tsukuba.ac.jp

SPECIFIC AIMS

Although a role for the renin-angiotensin system (RAS) in the regulation of erythropoiesis has long been suspected, its mechanistic basis has not been clearly defined. The aim of this study was to elucidate the following questions regarding the interrelationship between activation of the RAS and erythropoiesis: first, does chronic activation of the RAS physiologically modulate positive or negative erythropoiesis in vivo; second, which of the three angiotensin receptor subtypes, AT1a, AT1b, or AT2, mediates RAS-modulated erythropoiesis in vivo; and finally, do increased Epo levels, direct stimulation of erythroid progenitors, or both, play a principal role in RAS-modulated erythropoiesis?

PRINCIPAL FINDINGS

1. Positive effects of RAS activation on erythropoiesis
In 3-month-old male human renin (hREN) and human angiotensinogen (hAGT) double transgenic mice (THM: Tsukuba hypertensive mice), persistent erythrocytosis was observed with an 25% increase in hematocrit levels compared with wild-type (WT) C57BL/6 male mice (Fig. 1 A). Reticulocyte count in the peripheral blood at age three months was significantly increased in THM compared with that in WT mice (Fig. 1B ). The increment in reticulocyte number suggested that erythrocytosis was associated with excessive production of erythrocytes. Splenomegaly, a secondary event to the erythrocytosis, was observed in THM (Fig. 1C ). These data suggest that the erythrocytosis in THM was attributable to augmented erythrocyte production.

View larger version (41K): [in this window] [in a new window]   Figure 1. Hematocrit levels of mice and associated phenotypes. A) Hematocrit levels in the WT, THM, THM/AT1-KO, and AT1-KO mice at age three months. Symbols represent individual measurements and horizontal bars represent mean levels of each group. B) Peripheral blood was stained with methylene blue (right panels) and the reticulocyte number was determined by microscopy examination in each group. Values shown (left) are the mean ± SE. Panels are representative smears of peripheral blood in each group and arrows indicate methylene blue-stained reticulocytes. C) The ratio of spleen weight (mg) to body weight (g) was determined, and results are depicted as a histogram. Typical spleens from WT and THM are shown in the right panel. Scale bar, 10 mm. *P < 0.05, **P < 0.01, ***P < 0.0001 vs. WT. +P < 0.05, ++P < 0.01, +++P < 0.0001 vs. THM.

2. Major role of AT1a receptor in RAS-induced erythrocytosis
To genetically elucidate the receptor subtype responsible for the erythrocytosis, we generated transgenic mice (TgM) carrying both hREN and hAGT transgenes in the genetically disrupted background of one of the major Ang II receptors, angiotensin II type 1a receptor (AT1aR), named THM/AT1-KO. These mice chronically overproduced Ang II in the absence of AT1aR. As shown in Fig. 1A , the genetic deletion of AT1aR from THM restored hematocrit levels to near-WT levels. The increased reticulocyte number (Fig. 1B ) and splenomegaly (Fig. 1C ) were also ameliorated to normal in the THM/AT1-KO mice. These data thus suggest that chronic activation of the RAS causes reticulocytosis and splenomegaly as well as erythrocytosis mainly through AT1aR in vivo.

3. Dispensable role of AT1a receptor on bone marrow cells for erythrocytosis
It has been shown that the activated RAS influences erythropoiesis via two pathways: Ang II increases Epo production in vivo, and also directly stimulates proliferation of normal early erythroid progenitors through AT1 receptors in vitro. To examine whether the erythrocytosis observed in THM was due to increased production of Epo from kidney or liver cells or to the direct stimulation of early erythroid progenitors of bone marrow-derived cells, we conducted bone marrow transplantation experiments. If the erythrocytosis results from the accelerated proliferation of erythroid progenitor cells mediated through AT1aR on bone marrow cells, then hematocrit levels in THM transplanted with the marrows of AT1-KO mice (THM<-AT1-KO) should be decreased compared with those of THM transplanted with the marrows of WT mice (THM<-WT). Hematocrit levels at five weeks after bone marrow transplantation are shown in Fig. 2 A. In both THM groups transplanted with marrows of WT and AT1-KO, apparent erythrocytosis was observed compared with control WT mice transplanted with marrows of the WT mice (WT<-WT) group. Importantly, no significant difference was observed in hematocrit levels between the THM<-WT and THM<-AT1-KO groups. This result conclusively shows that AT1aR on bone marrow-derived cells is dispensable for the peripheral blood erythrocytosis induced by Ang II.

View larger version (10K): [in this window] [in a new window]   Figure 2. Hematocrit levels in bone marrow-transplanted mice and respective Epo levels. A) Hematocrit levels in mice at five weeks after bone marrow transplantation. Mean hematocrit levels are shown ± SE. The THM<-WT, for example, represents THM transplanted with bone marrow of WT mice. n = 8 for all groups. *P < 0.01 vs. WT<-WT. N.S., not significant. B) Plasma Epo level was measured in WT, THM, and THM/AT1-KO mice at age 3 months and the results are expressed as relative to that in WT mice (set at 100%). *P < 0.05, **P < 0.0001 vs. WT. +P < 0.05 vs. THM. C) Total RNA was isolated and erythropoietin expression was determined by quantitative real-time PCR. Data are indicated as the relative expression to that of average WT mice kidney Epo mRNA normalized for GAPDH (set at 100%). n = 6 for all groups. *P < 0.05 vs. WT.

4. Plasma Epo levels and quantification of kidney Epo mRNA
Our bone marrow transplantation results implied that the erythrocytosis induced by Ang II is mainly due to the stimulation of AT1aR present in the recipient organs. We therefore examined plasma Epo levels in each group of mice. As shown in Fig. 2B , THM showed a significant elevation of plasma Epo level compared with WT mice. In contrast, THM/AT1-KO mice showed a reduced Epo level compared with THM. To investigate whether the elevated plasma Epo levels result from increased Epo gene expression in the kidneys, the main source of Epo production in the adult stage, we examined kidney Epo mRNA of each group by quantitative real-time PCR. As shown in Fig. 2C , levels of Epo mRNA in THM kidney were 4.7-fold higher than those in WT mice kidney. Thus, our results demonstrated that the primary cause of the erythrocytosis observed in THM was the AngII-mediated overproduction of Epo, mainly through the stimulation of AT1aR on kidney cells.

CONCLUSIONS AND SIGNIFICANCE

An interrelationship between the RAS and erythrocytosis was first suggested by observations of patients in activated RAS conditions. Although a number of reports have suggested the involvement of the RAS in positive erythropoiesis, to our knowledge no clear in vivo experimental evidence for this issue had been presented to date. Here, the use of THM allowed us to demonstrate that chronic activation of the RAS had positive effects on erythropoiesis in vivo.

Our genetic manipulation has at least three advantages over administration of AT1 receptor antagonists or ACE inhibitors to THM. First, chronic and high-dose administration of ACE inhibitors or AT1-receptor antagonists is inevitably accompanied by drug toxicity, including hematopoietic toxicity. Second, rodents possess two AT1 receptor isoforms, designated AT1aR and AT1bR. By genetically deleting AT1aR from THM, we were able to verify the in vivo significance of AT1aR in blood pressure maintenance as well as erythropoiesis under conditions of a chronically activated RAS. Third, accumulating evidence suggests that Ang II-derived angiotensin fragments are biologically active and integral components of the RAS. Although it is conceivable that angiotensin fragment levels are augmented in THM, our genetic evidence proved that the receptor responsible for peripheral blood erythrocytosis in vivo is the AT1aR (Fig. 1A ).

RAS has been shown to influence erythropoiesis via two pathways as mentioned above. However, it is a great surprise that our bone marrow transplantation experiments clearly showed only a minor effect of the lack of AT1aR in bone marrow-derived cells on peripheral blood erythrocytosis in vivo (Fig. 2A ). Although the role of AT1aR on bone marrow-derived cells under pathophysiological and clinical conditions remains to be elucidated, our results demonstrate that the primary cause of erythrocytosis induced by chronic RAS activation is mediated by AT1aR on the recipient organ cells.

It is well established that, under physiological conditions, plasma Epo protein level shows an inverse relationship with hematocrit level. Our finding that THM with erythrocytosis exhibited elevated levels of Epo in kidney indicates that Ang II stimulated Epo production in THM, and suggests that the primary cause of the erythrocytosis observed in THM was the elevated plasma Epo levels from kidney cells (Fig. 2) .

Our novel findings indicate that 1) the systemic and persistent activation of RAS enhances erythropoiesis in vivo; 2) the major receptor subtype responsible for erythropoiesis in vivo is the AT1aR; 3)the lack of AT1aR in bone marrow-derived cells has a negligible effect on peripheral blood erythrocytosis; and 4) increased Epo levels via stimulation of AT1aR on kidney cells play a major role in erythrocytosis. Our results provide insight into the physiological relationship and clinical situation that exists between the RAS and erythropoiesis.

View larger version (17K): [in this window] [in a new window]   Figure 3. Schematic illustration of Ang II pathways in erythrocytosis in vivo

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3820fje;

This article has been cited by other articles:

				I. Kojima, T. Tanaka, R. Inagi, H. Kato, T. Yamashita, A. Sakiyama, O. Ohneda, N. Takeda, M. Sata, T. Miyata, et al. Protective Role of Hypoxia-Inducible Factor-2{alpha} against Ischemic Damage and Oxidative Stress in the Kidney J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1218 - 1226. [Abstract] [Full Text] [PDF]

				C. Gebhard, F. Petroktistis, H. Zhang, D. Kammerer, C. Kohle, K. Klingel, M. Albinus, C. H. Gleiter, H. Osswald, and A. Grenz Role of Renal Nerves and Salt Intake on Erythropoietin Secretion in Rats following Carbon Monoxide Exposure J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 111 - 116. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/14/2023 05-3820fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kato, H. Articles by Fukamizu, A. Search for Related Content PubMed PubMed Citation Articles by Kato, H. Articles by Fukamizu, A.