Angiotensin II infusion increases plasma erythropoietin levels via an angiotensin II type 1 receptor-dependent pathway

Kidney International, Vol. 60 (2001), pp. 83–86

Angiotensin II infusion increases plasma erythropoietin levels via an angiotensin II type 1 receptor-dependent pathway JAN GOSSMANN, RALF BURKHARDT, SEBASTIAN HARDER, TOMAS LENZ, ANNETTE SEDLMEYER, UTE KLINKHARDT, HELMUT GEIGER, and ERNST-HEINRICH SCHEUERMANN Funktionsbereich Nephrologie, Medizinische Klinik IV, Zentrum der Inneren Medizin, and Abteilung fu¨r klinische Pharmakologie, Zentrum der Pharmakologie, J.W. Goethe-Universita¨t, Frankfurt am Main, Germany

Angiotensin II infusion increases plasma erythropoietin levels via an angiotensin II type 1 receptor-dependent pathway. Background. Angiotensin-converting enzyme inhibitors (ACEIs) have been shown to lower hematocrit and erythropoietin (EPO), but a direct link between angiotensin II (Ang II) and EPO in humans has not been shown. Methods. Placebo or Ang II was infused for six hours in nine healthy male volunteers with and without blockade of the Ang II subtype 1 receptor (AT1R). EPO concentrations were measured 3, 6, 12, and 24 hours after the start of the infusion. Results. Ang II raised the mean arterial pressure by about 20 mm Hg. Consistent with the known diurnal variation, EPO levels rose significantly (P ⱕ 0.02) during the day in all groups. During Ang II infusion, EPO levels rose to significantly higher levels after 6 and 12 hours compared with placebo [9.9 ⫾ 3.5 vs. 7.2 ⫾ 3.1 mU/mL (3 h, P ⫽ NS); 16.9 ⫾ 4.5 vs. 8.8 ⫾ 3.7 mU/mL (6 h, P ⫽ 0.01); 17.0 ⫾ 8.6 vs. 11.1 ⫾ 4.7 mU/mL (12 h, P ⫽ 0.01)] and returned to baseline after 24 hours (7.9 ⫾ 3.8 vs. 10.6 ⫾ 8.6 mU/mL, P ⫽ NS). With AT1R blockade, blood pressure remained normal during Ang II infusion, and EPO levels were never significantly different from placebo [6.8 ⫾ 4.8, 10.5 ⫾ 5.6, 13.1 ⫾ 9.0, and 12.4 ⫾ 10.1 mU/mL at 3, 6, 12, and 24 h after infusion, respectively, P ⫽ NS]. Conclusions. Ang II increases EPO levels in humans. This increase requires the participation of AT1R.

erythropoietin (EPO) levels decreased during ACEI treatment [9–11], suggesting a link between angiotensin II (Ang II) and EPO. Furthermore, infusion with renin substrate in experimental animals has been shown to increase EPO concentrations [12]. Because the decrease in EPO concentrations was not statistically significant in all clinical studies on the use of ACEIs in post-transplant erythrocytosis [13–15], the influence of Ang II on EPO has been questioned [14]. We therefore performed a low-dose Ang II infusion in healthy volunteers with and without a blockade of AT1R to discover whether there was a stimulation of EPO production by Ang II and whether this could be inhibited by blockade of AT1R. METHODS Subjects After approval of the study protocol by the ethics committee of the J.W. Goethe-Universita¨t (Frankfurt/ Main, Germany) and written informed consent, nine healthy male volunteers (aged 29 ⫾ 4 years) were studied. The screening examination included history and physical examination, 12-lead echocardiogram, complete blood count, serum chemistry, and coagulation. All volunteers had normal blood pressure and body mass index. Blood pressure was monitored every five minutes and later at 30-minute intervals by the oscillometric method (Bosomat, Jungingen, Germany). Placebo (isotonic saline) or Ang II (Hypertensin, dissolved in isotonic saline; Novartis Pharma, Basel, Switzerland) was infused for six hours at a rate of 3 mL/hour. The experiments started at 7 a.m. Ang II was started at a dose of 1.25 ␮g/min, and the dose was increased in 0.625 ␮g increments until systolic blood pressure was approximately 10 mm Hg above baseline. The same individual Ang II infusion schedule was given after pretreatment of the volunteers with valsartan (Diovan; Novartis Pharma), 80 mg on the evening before and 80 mg on the morning four hours before the study. EPO plasma levels (Quantikine IVD;

A decrease of hematocrit or hemoglobin as a side effect of antihypertensive treatment with angiotensinconverting enzyme inhibitors (ACEIs) in patients with renal disease (patients on hemodialysis and renal transplant patients) has been described by several groups [1–5]. ACEIs therefore have become the standard treatment for postrenal transplant erythrocytosis (PTE) [6, 7], an increased production of red blood cells of unknown etiology in renal transplant recipients [8]. In most studies, Key words: anemia, ACE inhibition, hematocrit, hypertension, hemodialysis, transplantation, end-stage renal disease. Received for publication July 26, 2000 and in revised form January 16, 2001 Accepted for publication February 9, 2001

 2001 by the International Society of Nephrology

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Gossmann et al: Angiotensin II elevates EPO levels

Fig. 1. Mean arterial pressure in healthy volunteers during a six-hour placebo or angiotensin II (Ang II) infusion without and with pretreatment with valsartan, an Ang II subtype 1 receptor (AT1R) blocker. Symbols are: (䊐) placebo; (䊉) Ang II; (䉲) Ang II ⫹ valsartan; *P ⬍ 0.01 vs. placebo. Data are represented as mean ⫾ SEM.

R&D, Minneapolis, MN, USA) were measured 3, 6, 12, and 24 hours after the start of the infusion. Before and 24 hours after the start of the infusion, the hemoglobin concentration and reticulocyte counts were obtained. During Ang II infusion, one volunteer dropped out because of headache. One further volunteer did not complete the Ang II infusion after valsartan pretreatment because of problems with venous cannulation. Therefore, the number of subjects was nine for the placebo infusion, eight for the Ang II infusion, and seven for the Ang II infusion following AT1R blockade. Missing values were not replaced. Since calculation of the statistics using only the seven volunteers who completed the study did not affect the results, we decided to show the data strictly as they have been obtained. Data are given as mean ⫾ SEM. Statistical comparison was by paired Wilcoxon test and multivariate analysis of variance (ANOVA; SPSS 8.01; SPSS Inc., Chicago, IL, USA).

RESULTS Angiotensin II infusion increased mean arterial blood pressure significantly by approximately 20 mm Hg. This pressure response was completely inhibited by pretreatment with valsartan (Fig. 1), suggesting effective blockade of Ang II receptors. Compared with placebo, EPO levels were significantly higher after six hours of Ang II infusion and remained significantly elevated at 12 hours. Twenty-four hours after the start of the Ang II infusion,

Fig. 2. Erythropoietin (EPO) plasma levels in healthy volunteers following a six-hour placebo or Ang II infusion without and with pretreatment with valsartan, an AT1R blocker. Symbols are: (䊐) placebo; ( ) Ang II; ( ) Ang II ⫹ valsartan; *P ⫽ 0.01 vs. placebo. Data are represented as mean ⫾ SEM.

Table 1. Hemoglobin and reticulocytes in healthy volunteers before and 24 hours after placebo or angiotensin II (Ang II) infusion with and without Ang II subtype 1 receptor (AT1R) blockade

Hemoglobin g/dL at baseline after 24 hours Reticulocytes % at baseline after 24 hours

Placebo

Ang II

Ang II ⫹ valsartan

14.1 ⫾ 0.28 14.2 ⫾ 0.28

13.5 ⫾ 0.23 13.8 ⫾ 0.19

13.1 ⫾ 0.26 13.9 ⫾ 0.31

NS

11.1 ⫾ 1.34 8.6 ⫾ 1.02

9.4 ⫾ 1.28 10.6 ⫾ 1.40

10.4 ⫾ 1.09 11.0 ⫾ 1.20

NS

Pa

a

Compared to placebo, determined by paired Wilcoxon test. Data are represented as mean ⫾ SEM.

EPO concentrations returned to normal (Fig. 2). After pretreatment with valsartan EPO levels were never different from placebo following Ang II infusion. Consistent with the known diurnal variation of EPO levels, within-group comparisons showed a statistically significant (P ⱕ 0.02) increase of EPO levels over time in the placebo and in the Ang II ⫹ valsartan group. Analyzing the data by multivariate ANOVA, taking into account both time and treatment group, confirmed the data by showing a significant influence of both these variables on EPO concentrations. Hemoglobin concentrations and reticulocyte counts were not significantly altered by Ang II infusion (Table 1). DISCUSSION The discussion concerning the mechanism by which treatment with an ACEI leads to a decrease in hematocrit remains controversial. Most authors did [9–11], but some did not [13–15] find a significant decrease of EPO

Gossmann et al: Angiotensin II elevates EPO levels

levels in patients such treated. In earlier experiments in rats, an increase of EPO levels following infusion of renin substrate and a close correlation between renin and EPO levels have been demonstrated [12, 16, 17]. In humans, a recently published study showed that the increase in EPO following controlled hemorrhage could be augmented by infusion of Ang II [18]. For the treatment of PTE, both ACEIs and AT1R blockers have been used successfully [6, 19]. The pathophysiology of PTE remains unresolved. Some authors favored increased EPO production by the transplant or the native kidneys as a reason, whereas others have found no evidence for increased EPO levels in patients with PTE [8]. In our opinion, the effectiveness of ACEI and AT1R blocker treatment does not necessarily imply that increased EPO production must be the reason for PTE, but a lowering of EPO could still explain the decrease in hematocrit. Other mechanisms for a decrease in hematocrit during therapy with ACEIs or AT1R blockers have also been suggested. Inhibition of erythrocyte burst-forming units (BFU-E) proliferation in patients with PTE—but not in normal controls—by enalapril in vitro has been described, leading these researchers to suggest a direct pharmacological effect of the ACEI on red blood stem cells [20]. As another possible mechanism, an increase of a natural stem cell regulator in patients treated with captopril was found [21]. However, the finding that AT1R blockers also lower hematocrit argues against an effect of the ACEI itself [19, 22]. Mrug et al showed AT1R mRNA in erythroid progenitor cells after six days of culture [23]. The proliferative response to Ang II of these BFU-E, which occurred only from days 6 to 9 of culture, could be inhibited by losartan, suggesting that Ang II itself may have a direct effect on red cell proliferation. We now find that infusion of low doses of Ang II increases EPO levels, and this increase can be inhibited by AT1R blockade. It therefore appears likely that Ang II has an influence on EPO production, although the possibility of an Ang II-mediated decrease in EPO breakdown has to be considered. The exact physiological pathway by which Ang II influences EPO cannot be clarified by our study. Because Ang II infusion causes renal vasoconstriction, it is possible that decreased renal perfusion, rather than a direct effect of Ang II on EPO-producing cells, could be a mediator of the Ang II-induced EPO increase. The question of whether EPO-producing cells in renal interstitial tissue have receptors for Ang II has not been answered. These cells are not available for in vitro experiments, and double-labeling experiments for EPO and Ang II receptors have not been reported to date. Irrespective of the uncertainty regarding the details of the physiological pathway, we conclude that Ang II

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appears to have an influence on EPO levels in humans. With regard to the hematological side effects of ACEIs and AT1R blockers, an influence of these drugs on the proliferation of hematopoietic cells may also be involved. ACKNOWLEDGMENTS Angiotensin II (Hypertensin) and valsartan (Diovan) were kindly provided by Novartis Pharma. These data were presented in part at the 1999 Annual Meeting of the American Society of Nephrology (Miami, FL, USA). Reprint requests to Jan Gossmann, M.D., Funktionsbereich Nephrologie, Med. Klinik IV, Zentrum der Inneren Medizin, Klinikum der Johann Wolfgang Goethe-Universita¨t, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany.

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