Lack of angiotensin II-facilitated erythropoiesis causes anemia in angiotensin-converting enzyme-deficient mice

Abstract

While nephrologists often observe reduced hematocrit associated with inhibitors of angiotensin-converting enzyme (ACE), the basis for this effect is not well understood. We now report that two strains of ACE knockout mice have a normocytic anemia associated with elevated plasma erythropoietin levels. (51)Cr labeling of red cells showed that the knockout mice have a normal total blood volume but a reduced red cell mass. ACE knockout mice, which lack tissue ACE, are anemic despite having normal renal function. These mice have increased plasma levels of the peptide acetyl-SDKP, a possible stem cell suppressor. However, they also show low plasma levels of angiotensin II. Infusion of angiotensin II for 2 weeks increased hematocrit to near normal levels. These data suggest that angiotensin II facilitates erythropoiesis, a conclusion with implications for the management of chronically ill patients on inhibitors of the renin-angiotensin system.

Figure 1

Hematocrit. Tail vein blood was collected into microcapillary tubes and the hematocrit was determined for ACE.1 and ACE.2 knockout (KO), wild-type (WT), and heterozygous (HZ) mice. The number of mice in each group was as follows: ACE.1 KO, 12; ACE.1 WT, 13; ACE.1 HZ, 11; ACE.2 KO, 24; ACE.2 WT, 15; ACE.2 HZ, 14. The reduction in hematocrit between knockout and wild-type mice was highly significant, with P < 0.0001 for both knockout genotypes as compared with control mice. No significant difference was observed between wild-type and heterozygous mice. Data are presented as mean ± SE. RBC, red blood cell.

Figure 2

Plasma erythropoietin. Anesthetized mice were bled by cardiac puncture and plasma was immediately frozen. Erythropoietin levels were determined by radioimmunoassay. The number of mice in each group was as follows: ACE.1 KO, 10; ACE.1 WT, 9; ACE.1 HZ, 12; ACE.2 KO, 8; ACE.2 WT, 13; ACE.2 HZ, 9. The P value comparing ACE.1 knockout mice with wild-type mice is less than 0.01. A similar comparison of ACE.2 knockout mice with wild-type gave P < 0.05. Thus, the anemia present in ACE.1 and ACE.2 knockout mice was associated with elevated plasma levels of erythropoietin. Data are presented as mean ± SE.

Figure 3

Renal function. (a) Serum creatinine was measured from venous blood obtained from the tail. The number of mice in each group was as follows: ACE.1 KO, 11; ACE.1 WT, 12; ACE.1 HZ, 14; ACE.2 KO, 30; ACE.2 WT, 50; ACE.2 HZ, 50. (b) The creatinine clearance was measured as described in Methods. The number of mice in each group was as follows: ACE.1 KO, 5; ACE.1 WT, 6; ACE.1 HZ, 6; ACE.2 KO, 30; ACE.2 WT, 21; ACE.2 HZ, 19. All data are presented as mean ± SE. ACE.1 mice have a significant elevation of serum creatinine as compared with wild-type and heterozygous mice (P < 0.01). They also have a significant reduction of creatinine clearance as compared with these same control animals (P < 0.05). In contrast, ACE.2 knockout mice have no evidence of renal failure in that their serum creatinine and creatinine clearance values were not significantly different from wild-type littermate controls. While the creatinine clearance of ACE.1 and ACE.2 heterozygous mice is somewhat elevated above the levels of wild-type mice, these differences do not reach statistical significance. The difference in serum creatinine between ACE.1 wild-type mice and ACE.2 wild-type mice is probably due to the use of different reference laboratories to obtain these data.

Figure 4

Plasma angiotensin peptide levels. Anesthetized mice were bled by cardiac puncture and plasma was immediately frozen. Plasma angiotensin I and angiotensin II peptide levels were determined by radioimmunoassay. The number of mice in each group was as follows: ACE.1 KO, 12; ACE.1 WT, 10; ACE.1 HZ, 11; ACE.2 KO, 11; ACE.2 WT, 13; ACE.2 HZ, 10. (a) Both ACE.1 and ACE.2 knockout mice have a marked reduction of plasma angiotensin II, with P < 0.0001 compared with either wild-type or heterozygous mice. (b) Both ACE.1 and ACE.2 knockout mice have a marked reduction of the angiotensin II / angiotensin I ratio. This is due to a reduction of plasma angiotensin II and an elevation of plasma angiotensin I levels (see Table 1). While ACE.1 and ACE.2 heterozygous mice have normal levels of plasma angiotensin II (a), the elevation of angiotensin I present in these mice resulted in a significant reduction of the angiotensin II / angiotensin I ratio as compared with wild-type mice (P < 0.01). Data are presented as mean ± SE.

Figure 5

Acetyl-SDKP. Venous blood was obtained from the tail and plasma was immediately frozen. Acetyl-SDKP peptide levels were measured by radioimmunoassay. The number of mice in each group was as follows: ACE.1 KO, 7; ACE.1 WT, 6; ACE.1 HZ, 6; ACE.2 KO, 18; ACE.2 WT, 9; ACE.2 HZ, 10. Both ACE.1 and ACE.2 knockout mice have an elevation of plasma acetyl-SDKP as compared with wild-type or heterozygous mice. The highest levels of peptide were present in the ACE.1 knockout mice, animals completely null for ACE activity. Data are presented as the means ± SE.

Figure 6

Red cell mass. Whole blood was obtained by cardiac puncture from donor ACE.2 heterozygous mice. After labeling with ⁵¹Cr, an aliquot was infused via a carotid artery catheter. After allowing for equilibration, blood was obtained by cardiac puncture and used to determine total blood volume and total red cell volume. An implied plasma volume was then calculated. All data were normalized for the weight of the animal. The number of mice in each group was as follows: ACE.2 KO, 7; ACE.2 WT, 7. While the blood volume of ACE.2 knockout mice is equivalent to that of wild-type mice, the data show that the ACE.2 knockout mice have roughly a 25% reduction of red cell mass (P < 0.01). Data are presented as mean ± SE.

Figure 7

Angiotensin II infusion increases hematocrit. A cohort of wild-type and ACE.2 knockout mice were evaluated for systolic blood pressure and hematocrit. Animals were then implanted with osmotic minipumps delivering either angiotensin II (+ Ang) or vehicle (Control). After 2 weeks, blood pressure and hematocrit were reassessed. (a) The systolic blood pressure of the mice before and after angiotensin II infusion. The number of mice in each group was as follows: ACE.2 WT, 6; ACE.2 KO + Ang, 6; ACE.2 KO control, 4. Infusion of small amounts of angiotensin II raised the blood pressure of ACE.2 knockout mice to levels comparable with those of wild-type mice. (b) The hematocrits of the mice described in a were studied before and after angiotensin II infusion. ACE.2 knockout mice treated with angiotensin II showed a significant increase of hematocrit (P < 0.001) to levels near those of wild-type mice.